The present invention is directed to assays for detection of a thrombophilic disease in a patient. The assays, which are based on the diagnosis of the antiphospholipid antibody syndrome (aPL syndrome) in an individual, detect the capacity of a patient sample to inhibit the binding of annexin-V, an anticoagulant protein, to a phospholipid substrate, and thereby block the anticoagulant activity of annexin-V.
The presence of antibodies in blood which recognize anionic phospholipids, or anionic phospholipid protein complexes, has been associated with a thrombophilic syndrome known as the antiphospholipid (aPL) antibody syndrome. The aPL syndrome, which is also known as the lupus anticoagulant syndrome, or the anticardiolipin antibody syndrome, is characterized by arterial and venous thrombosis or, by recurrent pregnancy loss attributed to placental thrombosis (Lockwood C. J., Rand J. H., 1994, Obstet. Gyn. Survey 49: 432; Lockshin M. D., 1996, Lupus 5: 404; Shapiro S. S., 1996, Annu. Rev. Med. 47: 533; Asherson R. A, Khamashta M. A., Ordi Ros J., Derksen R. H. W. M., Machin S. J., Barquinero J., Outt H. H., Harris E. N., Vilardell-Torres M., Hughes G. R. V., 1989, Medicine 68: 366). The aPL syndrome thus comprises a thrombophilic disease distinguished by antibodies that recognize anionic phospholipid complexes. The disorder may occur spontaneously or in conjunction with another autoimmune disorder such as systemic lupus erythematosus (Conley C. L., Hartmann R. C., 1952, J. Clin. Invest. 31: 621), hence the name "lupus anticoagulant."
Paradoxically, aPL syndrome antibodies appear to manifest as "lupus anticoagulants" (Conley et al. 1952, J. Clin. Invest. 31: 621; Shapiro S. S., 1996, Annu. Rev. Med. 47: 533; Triplett D. A., 1996 Lupus 5: 43) in vitro by inhibiting phospholipid-dependent blood coagulation. Yet, in vivo, these "anticoagulants" are associated with thrombotic mechanisms, and rarely, if ever, with any bleeding disorders.
Evidence has been accumulating that the aPL syndrome bears relationship to the presence of anticoagulant proteins found on the surface of endothelial cells that come into contact with blood. Such anticoagulant proteins include a family of proteins known as annexins, a principal member being annexin-V. Annexin-V, which is also known as placental anticoagulant protein--1 or vascular anticoagulant--.alpha., has potent anticoagulant properties in vitro that are based on its high affinity for anionic phospholipids and its capacity to displace coagulation factors from phospholipid bilayers (Andree et al., 1992, in Andree (ed.) Maastricht, the Netherland, Universitaire pers Maastricht, p. 73). Annexin-V, which is normally present on the apical surface of placental syncytiotrophoblasts, has been found to be reduced on apical membranes of placental villi from aPL syndrome patients (Krikun et al., 1994, Placenta 15: 601; Sammaritano et al., 1992, J. Clin. Immunol. 12: 27).
The aPL syndrome has been shown to be an important risk factor for stroke, equivalent in predictive value to hypertension (Rand, J H, 1998, Am. J. Med. Sci. (In press)). Moreover, aPL antibodies are associated with recurrent arterial and venous thrombosis. Approximately 40% of patients on hemodialysis have aPL antibodies, which may be associated with graft thrombosis (the problem of graft thrombosis alone has an estimated cost in the United States of $700 million/year, which is equal to the cost of hemodialysis). aPL antibodies have also been associated with recurrent pregnancy loss, however, current diagnostic methods have not proven specific enough to allow for treatment before a woman has experienced at least three spontaneous abortions. Currently, it is recommended that patients diagnosed for aPL antibodies with thrombosis receive high intensity anticoagulant therapy for the remainder of their lives. Thus there is a need for developement of specific assays for diagnosis and monitoring of thrombophilic disease in susceptible individuals, i.e., those known to have aPL syndrome or at risk for aPL syndrome.
Such methods can be useful for screening for risk of stroke, recurrent miscarriages and risk of thrombosis or embolism, as well as evaluating for the etiology of patients presenting with the above disorders, all of which may be classified as thrombophilic diseases. In addition, there has been an increasingly frequent clinical problem in the identification of patients with incidental positive antiphospholipid antibody tests (7-10% of the general population, and up to 40% of ill hospital patients) with no accepted means to distinguish abnormalities that are clinically relevant (disease related) from those that are irrelevant. The present invention is directed to providing specific assays to be used as a basis for making those distinctions.
The methods of the present invention are based upon the interaction of phospholipid substrates, aPL antibodies and annexin-V and, also, the consequent coagulant activity of the complex exposed to clotting factors. The methods can be used to diagnose thrombophilic disease (or hypercoaguable disease) in susceptible individuals (i.e., those having or at risk of developing aPL syndrome) by utilizing the reduction in binding of annexins, particularly annexin-V, to phospholipid substrates in the presence of a blood specimen from a known or suspected aPL syndrome patient as a marker of the disease. The present invention thus provides for specific assays which are predictive of and can monitor thrombophilic disease, as well as the success of treatment.